39
RESULTS AND DISCUSSION
RESULTS AND DISCUSSION
In this experimental part the formation of selected supramolecules and of the nano-gold will be studied using:
Mass spectrometry and capillary electrophoresis.
The study of supramolecules will be done on cucurbiturils, important macrocycles.
Mass spectrometry will concern formation of gold clusters and their interactions with cucurbiturils and anti-alzheimer drug, Huperzine A.
Capillary electrophoresis was applied to study the formation of gold nano-particles.
41
1. MALDI TOF MS OF SUPRAMOLECULAR COMPLEXES
Intention of this part of work was to develop analytical method (capillary electrophoresis) for separation of cucurbiturils CB[n], n = 5, 6, 7, 8, important macrocyclic compounds. However, up to now the separation was studied only by Dheba M. Abobaker (*), but the separation of CB[n]’s was not reached. Capillary electrophoresis of cucurbiturils separation was not published up to now. Therefore, in this work we were trying to search for some other compounds forming supramolecules, which might enable CB separation.
In the first part the formation of supramolecules will be followed by mass spectrometry.
1.1. Mass spectra of cucurbituril[6] and formation of some supramolecular complexes.
In this part of work the formation of supramolecular complexes between CB[6], and some other molecular will be studied. CB[6], empirical formula C36H36N24O12 and molecular weight is 996.29 g/mol.
Preparation of samples:
Stock solution of 0.05 mM of CB[6] was prepared in 1.0 mM of NaCl, because the compound is insoluble in water. The mass spectra were measured using an Axima-CFR mass spectrometer (Manchester, U.K.); controlled by Crates Kompact V5.2.0. Launchpad 1.2.0 software was used for data acquisition. The Axima-CFR mass spectrometer was equipped with a nitrogen laser (Wavelength 337 nm).
In order to measure the mass spectra, 1 µl of 0.05 mM of CB[6] was applied onto the MALDI target and dried in stream of air, then inserted into the mass spectrometer. Mass spectrometry of CB was done in two different ways. Either using Laser Desorption Ionization (LDI) or using matrices (MALDI).
---
(*) Dheba M. Abobaker, Diploma Thesis, Some Application of Capillary Zone Electrophoresis The use of supramolecular interaction in CE.
Results:
LDI results showed that no peaks of such species as {CB[6].2H}+ or [M+H]+ were observed however, peaks corresponding to {CB[6].Na.H}+ were observed. Comparison of theoretical and experimental spectra concerning {CB[6].Na.H}+ are shown in Fig. 12 A, B.
Fig. 12. (A) Theoretical model of {CB[6].Na.H}+, (resolution 2000), and (B) experimental mass spectrum concerning {CB[6].Na .H}+
M/Z
Rela ti v e I nt ens it y [ % ]
BM/Z
Relative Intensity [%]
A
43 1.1.1. Study of supramolecular complex formation of some simple compounds with
CB[6]
In this part of the work interaction of simple organic molecules with CB[6] will be studied following mass spectra of some simple compounds in the mixture with CB[6].
1.1.1.1. Interaction of CB[6] with 1,10-phenanthroline
1,10-phenanthroline is a white crystalline compound, molecular formula C12H8N2, molecular weight 180.30 g/mol. Chemical structure of 1,10- phenanthrolin is shown in Fig.
13. The compound is easy to be protonated to phenanthrolinium cation (C12H8N2.H+).
Preparation of samples:
Stock solution of 1,10-phenanthroline 1 mM was prepared by dissolving 0.18 mg of 1,10-phenanthroline in distilled water. In order to measure the masss spectra, 1 µl of 1 mM 1,10-phenanthroline was applied onto the MALDI target. Mixtures of CB[6] and 1,10- phenanthroline were prepared, by mixing 1 µl of 0.05mM CB[6] with 1 µl of 1 mM 1,10- phenanthroline, in the ratio (1:1). Then 1 µl of mixture was taken and applied onto the spot of MALDI target and mixed with α-cyano-4-hydroxy cinnamic acid (CHC) as matrix.
After drying the samples by stream of air at room temperature, then inserted into MALDI instrument, the measurement was done when the vacuum reached to 1.2 E-4 Pa, and linear positive mode was chosen.
Results:
According to MALDI mass spectra, there is no interaction between CB[6] and 1,10- phenanthroline, probably because of small size of the CB[6] cavity. Laser Desorption Ionisation (LDI) spectra of 1,10-phenanthroline are shown in Fig. 14 A, B.
Fig. 13. Chemical structure and computer model of 1,10-phenanthroline.
Fig. 14. Comparison of experimental (A), and theoretical spectra (B) of 1,10-phenanthrolinium ion [C12H8N2 .H]+ (Resolution 1000)
1.1.1.2. Interaction of CB[6] with 1,1’-dimethyl-4,4’-bipyridinium dichloride.
1,1-Dimethyl-4,4-bipyridinium dichloride is also known as paraquat dichloride or methyl viologen (MV)+2. The compound is one of the most widely used herbicides in the world. Methyl viologen (MV)+2 is a white powder and has the molecular formula [C12H14N2]2+.2Cl, molecular weight 257.16 g/mol. Chemical structure and computer model were present in Fig. 15.
M/Z
M/Z
Relative Intensity [%] Relative Intensity [%]
A B
45 Sample preparation:
Stock solution of methyl viologen was prepared by weighting 0.1285 mg and dissolved in distilled water to get 0.5 mM stock solution, then 1 µl of 0.5 mM was mixed with 1 µl of saturated solution CHC matrix onto one spot of the MALDI target. And, 1 µl of sample was mixed with 1 µl of 0.05 mM CB[6], and 1 µl of CHC were deposited separately on the target. After drying the samples by stream of air at room temperature, then inserted into MALDI instrument, the measurement was done when the vacuum reached to 1.2 E-4 Pa, and linear positive mode was chosen.
Results:
Mass spectra of a mixture of CB[6] and methyl viologen are shown in Fig. 16 A, B, C, and D, E. The Fig. 16 A, B show the comparison of theoretical and experimental spectra of methyl viologen. In Fig. 16 C, the mass spectra shows that there is small peak which indicates the complex formation of [CB[6].MV]+. Other species of [CB[6].H.MV]+ refer to the complex of methyl viologen with protonated CB[6]. Theoretical model of [CB[6].MV]+ and [CB[6].MV.H]+ are shown in Fig. 16 D, E.
Fig. 15. Chemical structure and computer model of methyl viologen.
Fig. 16. Comparison of experimental (A), and theoretical spectra (B) of methyl viologen MV 2+ (Resolution 500).
Fig. 16. (C) Experimental spectrum showing the simultaneous formation of [CB[6].MV.H]+ and [CB[6].MV]+ ions.
Relative Intensity [%]
Relative Intensity [%]
M/Z
M/Z
A B
[CB[6].MV]+
[CB[6].MV.H]+
M/Z
Relative Intensity [%]
C
47 Fig. 16. (D) Theoretical model of [CB[6].MV.H]+ and theoretical model of
[CB[6].MV]+ ions (E), (Resolution 2000).
1.2. Mass spectra of cucurbituril[8] and the formation of some supramolecular complexes.
Cucurbituril[8], empirical formula C48H48N32O16, molecular weight 1328.39 g/mol.
Preparation of sample:
Stock solution of 0.05 mM of CB[8] was prepared in 1.0 mM of NaCl. In order to measure the mass spectra. 1 µl of 1 mM solution of CB[8] was applied onto the MALDI target and dried in stream of air then inserted into the mass spectrometer.
Results:
During LDI experiments, no peaks of such species as CB[8]+ or CB[8].H+ were observed in the mass spectra. However, the signal corresponding to [CB[8].Na]+ was observed at 1351.0 Da (Fig. 17 A, B).
Relative Intensity [%]
Relative Intensity [%]
M/Z
M/Z
D E
Fig. 17. Comparison of experimental (A) and theoretical model (B) of {CB[8].Na}+ mass spectrum (Resolution 2500).
Relative Intensity [%]
M/Z
A
Relative Intensity [%]
M/Z
B
49 1.2.1. Interaction of CB[8] with 1,10-phenanthroline
CB[8] cavity is bigger than that of CB[6], so we have examined the possibility to insert 1,10-phenanthroline into that cavity.
Preparation of sample:
Stock solution of 0.05 mM of CB[8] was mixed with 1,10-phenanthroline (1 mM ) in the ratio 1:1 v/v, and saturated solution of CHC in acetonitrile was used as matrix.
Results:
The mass spectra obtained are shown in Fig. 18 A, B. The peak at m/z 1510.1 may be due to the formation of the supramolecular complex with 1,10-phenanthroline.
Fig. 18. (A) Mass spectrum concerning the mixture of (1:1) CB[8] and 1,10- phenanthroline {CB[8].C12H8N2.H}+ and theoretical model (B) of {CB[8] C12H8N2 .H}+ (Resolution 1000).
1.2.2. Interaction of CB[8] with 1,1’-dimethyl-4,4’-bipyridinium dichloride
Stock solution of 0.5 mM methyl viologen (MV2+) was prepared by weighting 0.1285 mg and dissolved in distilled water, then 1 μl of solution was mixed with 1 μl saturated solution of CHC matrix onto a spot of MALDI target. Separately on the other spot, 1 μl of (MV2+) was mixed with 1 μl of 0.05 mM CB[8] in the ratio (1:10 v/v), and 1 μl of CHC. After drying the samples by stream of air, the target was inserted into MALDI instrument.
The mass spectra are shown in Fig. 19 A, B. The signal at 1514.8 may be related to the fact that CB[8] is forming the supramolecular complex with methyl viologen {CB[8].MV}+.
B
Relative Intensity [%]
M/Z
51 Fig. 19. (A) Mass spectra concerning the complex formation of (1:10) {CB[8].MV}+
and theoretical model (B) of {CB[8].MV}+ mass spectrum.
Relative Intensity [%]
Relative Intensity [%]
M/Z
M/Z
A B
2. MASS SPECTROMETRY OF GOLD CLUSTERS
An attempt has been done to analyze also nano gold solutions (prepared by gallic acid) using mass spectrometry.
Sample preparation
Nano gold solution was prepared by adding 1.5 ml HAuCl4 (1 mM) to 1.5 ml of distilled water and 0.5 ml of 0.5 mM gallic acid. The color of solution rapidly changed from colorless to red. The total concentration of gold was 0.4 mM and 0.07 mM gallic acid.
Mass spectrometry of nano gold
The mass spectra were measured using an Axima-CFR mass spectrometer (Manchester, U.K.); controlled by Kratos Kompact V5.2.0. Launchpad 1.2.0 software was used for data acquisition. The Axima-CFR mass spectrometer was equipped with a nitrogen laser (Wavelength 337 nm). In order to measure the spectra, 1 µl of nano gold solution was applied onto the MALDI target, dried in stream of air at room temperature and inserted to mass spectrometer. The measurement was done when the vacuum reached to 1.2 E-4 Pa.
An example of mass spectra of nano gold by Laser Desporption Ionization (LDI) mode is given in Figure 20 A, in the range m/z 190-700 Dalton.
It is evident from this figure that single charged of Aun+ clusters (n = 1-3) are formed.
In Figure 20 B, it is shown that in the range 1000-5000 Dalton clusters of gold Aun+(n =5-25) are formed. It is interesting to note that intensity of clusters with even number of gold atoms are lower than those for odd numbers. However, it is difficult from mass spectra to conclude what is real composition of nano gold clusters as they can be partially decomposed by laser during laser desorption ionization (LDI).
The results are in agreement with observations in the literature, for example [44].
53 Fig. 20. (A) LDI mass spectrum showing the formation of charged gold clusters
Aun+ (n = 1-3). Conditions: linear positive ion mode.
Fig. 20. (B) LDI mass spectrum showing the formation of high gold clusters Aun+ (n = 5-25). Conditions: linear positive ion mode.
0 20 40 60 80 100
1000 2000 3000 4000 5000
M/Z Au5+
Au24+ Au6+
Au7+ Au9+
Au10+ Au8+
Au11+ Au12+
Au13+ Au14+
Au15+ Au17+
Au18+ Au19+
Au20+ Au21+
Au25+ Au22+
Relative Intensity [%]
B 0
20 40 60 80 100
200 300 400 500 600 700
M/Z Au1+
Au3+
Relative Intensity [%]
Au2+
A
3. MASS SPECTROMETRY OF HUPERZINE A
Huperzine A (Hup A) is an alkaloid isolated from the Chinese plant Huperzia serrata [45]. Huperzine A, a reversible acetyl cholinesterase inhibitor for the treatment of Alzheimer disease, empirical formula C15H18N2O, molecular weight 242.32 g/mol and its molecular structure can be seen in Figure 21.
Fig. 21. Chemical structure of Huperzine A (Hup A).
As it is described in the literature Hup A interacts with nanogold [46]. We have also studied mass spectrometry of Hup A-nanogold with the future plan to use Huperzine A as an additive during the electrophoretic separation of nanogold clusters.
Sample preparation
Stock solution of Hup A was prepared by dissolving 6.2 mg of compound in 2.5 ml of distilled water and then 25 μl of 1M HCl was added, the total concentration of Hup A was 10 mM.
Mass spectrometry of Hup A
The mass spectra were measured using an Axima-CFR mass spectrometer (details are given in page 52). In order to measure the spectra, 1 µl of Hup A solution was applied onto
55 the MALDI target, dried in stream of air at room temperature and inserted to mass spectrometer. The measurement was done when the vacuum reached to 1.3 E-4 Pa.
An example of mass spectrum of Hup A is given in Figure 22 A. It is clear that we are observing single charged of [HupA. H]+peak. This result is in a good agreement with computer model (Fig. 22 B).
Figure 23 also shows that except [HupA. H]+ there is a formation of [HupA. Na]+ adduct and the formation of the dimmer of Hup A was also found [(HupA)2. H]+ and [(HupA)2. Na]+, in agreement with the literature [47].
Fig. 22. Mass spectrum of experimental (A) [HupA. H]+, and the theoretical model (B) of [HupA. H]+ ion (Resolution 1000). Conditions: 10 mM HupA, linear positive ion mode
Fig. 23. Mass spectra concerning the formation of [HupA. Na] +and [(HupA)2. H]+. Conditions: 10 mM Hup A, linear positive ion mode.
Relative Intensity [%]
[HupA. H]+
[(HupA)2.Na]+
[(HupA)2.H]+ [HupA. Na]+
0 20 40 60 80 100
250 300 350 400 450 500
M/Z
243.01
507.52
265.03
508.60 485.42
245.06
57
4. MASS SPECTROMETRIC STUDY OF THE INTERACTION OF NANO GOLD WITH HUPERZINE A
Because it is known that nanogold can interact with amino group, in this chapter we have followed by mass spectrometry the interaction of Huperzine A with nanogold.
1 µl of nanogold solution prepared as described above was applied onto the MALDI target and mixed with a diluted Huperzine A aqueous solution and the mixture was analyzed after dried in an air stream at room temperature.
An example of LDI mass spectrum concerning a mixture of nanogold-Hup A is given in Figure 24.
Fig. 24. Mass spectra concerning the interaction of Huperzine A with nanogold forming [HupA. Au]+and [(HupA. Au3]+. Conditions: linear positive ion mode.
0 10 20 30 40 50 60 70 80 90 100
450 500 550 600 650 700 750 800 850 900
M/Z
590.71
680.76
832.40 438.80
[HupA ∙ Au]+ Au3+
[HupA ∙ Au2Na2]+
[HupA ∙ Au3]+
Relative Intensity [%]
878.20
[HupA. Au3.Na2]+
It follows from Figure 24 that really Hup A is interacting with nanogold and that in the m/z range 400-900 Dalton there are several peaks observed.
Through the analysis of the mass spectra it was concluded that there is a formation of an adduct HupA: Au = 1:1, but also the formation of HupA.Au2 and HupA.Au3 adducts were observed. Adducts of HupA.Au2 and HupA.Au3 a containing two sodium cations are present.
The scheme of possible interaction of Huperzine A with nanogold is given in Figure 25.
Fig. 25. The scheme of possible interaction of Huperzine A with nanogold.
+
Au
NH2Au
NH2NH2 HUP A
HUP A
59 Conclusions:
Found interaction of Huperzine A with nanogold can be important for several reasons:
➢ Nanogold can serve as Huperzine carrier and in this way the distribution of the drug in human body might be changed.
➢ Nanogold-HupA adduct might show increased activity contra Alzheimer disease.
However, it would be necessary to make corresponding experiments.
➢ Interaction of Hup A with nanogold can also be used to stabilize nanogold solutions.
Such stabilization of nanogold can have practical importance.
➢ Derivatization of Hup A with nanogold can also be used to increase the sensitivity of Hup A determination. It can be suggested that the determination of Hup A might be much more sensitive, either in mass spectrometry, but also in capillary electrophoresis.
➢ Last, but not least, Hup A can be also used as additive in capillary electrophoresis to follow its effect on the separation of nanogold clusters.
As consequence of our results, we aimed to focus the study in the last mentioned items, but for some technical reasons this part of the work was not completed.
5. CAPILLARY ELECTROPHORESID OF GNP
Gold nano-particles (GNPs) were prepared from HAuCl4 using several reducing agents such as hydrogen peroxide, hydrazine, citric and gallic acids.
5.1. Chemicals and products
Gold (III) chloride trihydrate (AuCl3.3H2O) and gallic acid were purchased from Sigma-Aldrich (Stenheim, Germany). Sodium hydroxide was obtained from Merck (Darmstadt, Germany). Hydrogen peroxide and acetone were prepared from Penta (Prague, Czech Republic,) and sodium tetraborate (Na2B4O7.10H2O) was obtained from Lachema (Brno, Czech Republic)
Double distilled water obtained from the quartz distillation stand of Heraeus Quartzschmelze (Hanau, Germany) was used to prepare all the solutions. Electrolyte solutions were filtered through a glass crucible S4 filters from Kavalier (Sázava, Czech Republic).
5.2. Equipment and capillary electrophoretic conditions
The CE experiments were carried out on a SpectraPhoresis 2000 Thermo separation products (Fremont, CA, USA), which was driven by the CE software (version 3) operating under IBM OS/2 (version 1.2) equipped with a UV-Vis detector containing a programmable high-speed scanning multiple wavelength detector. The separation of GNP was done in an uncoated fused–silica capillary which was purchased from Watrex (Prague, Czech Republic), the total length of the capillary was 45.5 cm (38.5 cm to the detector) and internal diameter 75 µm. Burning off the polyamide coating on the capillary created a detection window. The detection wavelength was 190, 200, 320 nm. The detection was done at the cathode side. Before the separation of GNP, the capillary was washed with 0.1 M NaOH for 5 min, followed by washing with distilled water for 5 min, then with run buffer for 5 min as a buffer was used usually 20 mM borate (pH 9.2), at 25˚C, if not otherwise stated. Between each run and at the end of the day the capillary was washed with distilled water for 2 min and then with buffer for 2 min at 25˚C.
The EOF was measured using either acetone or mesityl oxide (1%) as neutral
61 markers. An example of the EOF determination is given in Figure 26. It follows from this figure that the migration time of acetone was 4.37 min, which is the normal value for quartz capillary and alkaline pH (9.2).
Fig. 26. Determination of EOF.
Conditions: 20 mM borate buffer pH 9.2, applied voltage 10 kV, EOF marker (acetone 1%), hydrodynamic injection 1 s, UV detection at 190 nm, and temperature 25 0C.
) O2
(H2
using hydrogen peroxide . Synthesis of GNPs
3 . 5
Gold nanoparticles were synthesized by adding 100 μl of 16 mM HAuCl4 to 1400 μl of distilled water and 50 μl of 30 % H2O2 (9.8 M), 100 μl of 0.1M NaOH and 50 μl of 1%
acetone. The color of solution rapidly changed from colorless to different blue color according to the concentration of nanogold.
The overall reaction is suggested as for example:
2 AuCl4- + 3 H2O2 = 2 Au + 3 O2 + 8 Cl- + 6 H+
However, the reaction depends strongly on pH (Fig. 27).
EOF
-0.002 0 0.002 0.004 0.006 0.008 0.01
0 1 2 3 4 5 6 7 8 9 10
Migration time, min
Absorbance, mAU
-0.002 0 0.002 0.004 0.006 0.008 0.01
0 1 2 3 4 5 6 7 8 9 10
Migration time, min
Absorbance, mAU EOF
Fig. 27. Distribution diagram of Au3+ species in chloride solution as function of pH.
5.3.1. Separation of gold nanoparticles
Even it in the literature there are several references concerning capillary electrophoresis of nanogold for example [48, 49, 50], the electrophoretic behavior of GNPs is still not well explained and not fully understood. For example, the charge of GNPs in aqueous solution is negative [51], but it is still not explained well why it is. Therefore, the aim of this work was to study the electrophoretic behavior of GNPs and to contribute to this field.
An example of the electropherogram concerning the separation of a sample of nanogold prepared using hydrogen peroxide is given in Figure 28. It follows from this figure that EOF is at about 4.3 min. and that at much longer time (about 7.3 min) we are observing three peaks. Peak 1 after EOF has been identified as H2O2 peak. The explanation why this peak of neutral H2O2 is slightly moved after EOF is that at this pH (9.2), the hydrogen peroxide is partially dissociated into HOO- anion ( H2O2 = HOO - + H+, pK ≈ 11.6).
Electropherogram of 1 % hydrogen peroxide is shown in Figure 30.
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3(Cl-) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3(C ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3(Cl-) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3( c ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
2 4 6 8 10 12
0.0 0.2 0.4 0.6 0.8 1.0
Fraction
pH
A u ( O H ) 3 A u ( O H )4−
A u C l 3O H− A u C l 4−
A u ( O H ) 3(C ) [ A u 3+]
TOT = 1 0 . 0 0 M [ C l−
]TOT= 1 0 0 . 0 0 m M
63 Fig. 28. Electropherogram of GNPs prepared using H2O2.
Conditions: 0.96 mM HAuCl4, 297 mM H2O2, 6.06 mM NaOH, 50 μl of acetone (1%), 20 mM borate buffer pH 9.2, applied voltage 10 kV, hydrodynamic injection 20 s, UV detection at 190 nm, and temperature 25 0C.
As observed in Figure 28, 31 and 32, the peaks 2, 3, 4 are evidently corresponding to nano- gold, and the appearance of these peaks means that nano-gold prepared in the way as described above is containing nano-gold particles of three different sizes.
Species of gold corresponding to peaks 2, 3, 4 are migrating with much higher migration time than EOF. Evidently, the nano-particles of gold are negatively charged. The charge is probably generated by the adsorption of negative ions (in this case chloride), perhaps partly also hydroxide. Probable structure of gold nano particles with Cl- or OH- anion is given in Figure 29.
-0.005 0 0.005 0.01 0.015 0.02 0.025 0.03
0 1 2 3 4 5 6 7 8 9 10 11 12
Migration time, (min)
Absorbance (mAU)
EOF
1
2 3
4
Fig. 29. Scheme of GNPs possible structure.
A question arises if the chloride is just adsorbed or chemically bonded to nano-gold.
Sometimes in the literature it is assumed that not all gold atoms in the gold cluster (gold nano- particle) are reduced to zero and the presence of Au (I) was assumed [52]. In such a case of Au (I) presence there is a strong interaction of chloride with Au (I) as AuCl complex is extremely stable, log β(Au(I)Cl) = 10.64, and such a high value is a strong indication that some Cl- can be bound to some Au (I) which according to the literature can be present in nano gold clusters.
On the other hand log β(Au(III)Cl) = 1.70, which is 8 orders of magnitude lower.
Fig. 30. Electropherogram concerning a solution of 1% H2O2.
Conditions: 20 mM borate buffer pH 9.2, applied voltage 10 kV, hydrodynamic injection 5 s, UV detection at 190 nm, and temperature 25 0C
-0.005 0 0.005
0.01 0.015 0.02 0.025
0 2 4 6 8 10
Migration time, min
Absorbance, mAU
-0.005 0 0.005
0.01 0.015 0.02 0.025
0 2 4 6 8 10
Migration time, min
Absorbance, mAU
-0.005 0 0.005
0.01 0.015 0.02 0.025
0 2 4 6 8 10
Migration time, min
Absorbance, mAU
EOF
-0.005 0 0.005
0.01 0.015 0.02 0.025
0 2 4 6 8 10
Migration time, min
Absorbance, mAU
EOF
-0.005 0 0.005
0.01 0.015 0.02 0.025
0 2 4 6 8 10
Migration time, min
Absorbance, mAU
-0.005 0 0.005
0.01 0.015 0.02 0.025
0 2 4 6 8 10
Migration time, min
Absorbance, mAU
-0.005 0 0.005
0.01 0.015 0.02 0.025
0 2 4 6 8 10
Migration time, min
Absorbance, mAU
EOF
H
2O
265 5.3.2. The effect of the nano-gold solution aging
The nano-gold particles prepared by hydrogen peroxide were measured at different times and at different voltage (each 30 min, at 10, 15 kV). The results are shown in Figure 31, 32.
Fig. 31. Electropherograms showing the separation of GNPs measured at different times, (A) after mixing, (B) after 30 min, and (C) after 60 min. Conditions: 0.96 mM HAuCl4, 297 mM H2O2, 6.06 mM NaOH, 50 μl of acetone (1%), 20 mM borate buffer pH 9.2, applied voltage 10 kV, hydrodynamic injection 20 s, UV detection at 190 nm, and temperature 25 0C.
-0.005 0 0.005 0.01 0.015 0.02
0 1 2 3 4 5 6 7 8 9 10 11 12
Absorbance (mAU)
1
2 3
4
C
-0.005 0 0.005 0.01 0.015 0.02
0 1 2 3 4 5 6 7 8 9 10 11 12
Absorbance (mAU)
-0.005 0 0.005 0.01 0.015 0.02 0.025 0.03
0 1 2 3 4 5 6 7 8 9 10 11 12
Migration time (min)
Absorbance (mAU)
A B
1 1
2 3
3
4 4
2 EOF
Fig. 32. Electropherograms showing the separation of GNPs measured at different times, (A) after mixing, (B) after 30 min, and (C) after 60 min. Conditions: 0.96 mM HAuCl4, 297 mM H2O2, 6.06 mM NaOH, 50 μl of acetone (1%), 20 mM borate buffer pH 9.2, applied voltage 15 kV, hydrodynamic injection 20 s, UV detection at 190 nm, and temperature 25 0C.
-0.004 0 0.004 0.008 0.012
0 1 2 3 4 5 6 7 8
Absorbance (mAU)
-0.004 0 0.004 0.008 0.012
0 1 2 3 4 5 6 7 8
M igration time (min)
Absorbance (mAU)
A B
EOF 1
1
2 3
4 2
3
4
-0.004 0 0.004 0.008 0.012
0 1 2 3 4 5 6 7 8
Absorbance (mAU)
1
2 3
4
C
67 The first peak in Figure 31, 32 just, after EOF, was identified as H2O2. The peaks 2, 3, 4 are most probably peaks of nano-gold. We can see that the migration time of these peaks is increasing after some time and the resolution of nano gold peaks is better after 60 min. The explanation of this observation might be that the gold nano-particles are growing and aggregating with time, and their size is becoming bigger and bigger. That is why they are moving more slowly and so their migration time is higher.
5.4. Synthesis of GNPs using gallic acid
Gallic acid is a poly-phenolic compound that may be used as reductant, which is obtained from the hydrolysis of natural plant poly-phenols. We have used it as reductant to prepare gold nanoparticles by reducing HAuCl4 at room temperature [53]. A proposed reaction scheme for the reduction of HAuCl4 by gallic acid is given below:
Gold nanoparticles were synthesized by adding 1.5 ml HAuCl4 (1 mM) to 1.5 ml of distilled water and 0.5 ml of 0.5 mM gallic acid. The color of solution rapidly changed from colorless to different red or blue depending on gallic acid concentration. Figure 33 A shows the photographs of four solutions of nanogold prepared using different concentration of gallic acid and using different molar ratio of gallic acid to gold.
Also we prepared the same solutions of nano gold using the same concentration of HAuCl4 and gallic acid and we added 100 μl of 0.1 M NaCl to see the effect of Cl- on nano gold. We observed that the color of nano gold solutions is a little different, and after few days we found that the solutions of nano gold which containing amount of chloride are precipitated faster than the nano gold solutions without NaCl.
COOH
OH HO OH
3 2 AuCl4 2 Au
COO
O O
3
Photographs of nano gold solutions containing 100 μl of 0.1 M NaCl are shown in Figure 33 B.
Fig. 27. Photographs of nanogold prepared using different concentration of gallic acid (0.07, 0.05, 0.03, 0.04 mM) with molar ratio of gallic acid vs gold equal to 0.2, 0.13, 0.08, and 0.1, (A) no NaCl, and (B) 100 μl of 0.1 M NaCl.
Both, nano gold prepared using excess of Au (III) and excess of gallic acid were examined. An example of the electropherogram concerning experiments with excess of Au (III) is shown in Figure 34.
5 6 7 8
B
1 2 3 4
A
69 -0.0005
0 0.0005 0.001 0.0015 0.002 0.0025
0 2 4 6 8 10 12 14
Migration time, min
Absorbance
-0.0005 -0.0001 0.0003 0.0007 0.0011
0 2 4 6 8 10 12 14
Migration time, min
Absorbance
